Viability of bacterial dried cells

ABSTRACT

PCT No. PCT/GB94/01556 Sec. 371 Date May 12, 1995 Sec. 102(e) Date May 12, 1995 PCT Filed Jul. 19, 1994 PCT Pub. No. WO95/03395 PCT Pub. Date Feb. 2, 1995The viability of bacterial cells after drying is improved by aging the culture in the stationary phase for periods of around six hours prior to drying. Further improvement may be obtained by culturing in a nitrogen deficient medium. The bacterial cells accumulate trehalose to protect the cells against drying out and other cell damage and the medium is further defined as having a higher than normal osmotic potential.

This invention relates to processes for production of a biologicalcontrol agent.

Various biological organisms (including bacteria and fungi) are known topossess general or specific anti-bacterial, anti-fungal (includinganti-yeast), insecticidal and/or herbicidal activity. Such organismshave enormous potential as alternatives or additions to chemicalagrochemicals, and are known as biological control agents or BCAs.Numerous naturally-occurring or genetically modified biological controlagents are known, and more may be discovered or developed in the future.These BCAs may be mass-produced by means of large-scale fermentation.

An obstacle to the successful commercial exploitation of biologicalcontrol agents (BCAs) is their formulation, which ideally involvesdesiccation. Drying bacteria (such as a Pseudomonas fluorescens BCA) ina commercial situation leads to losses in viability which can exceed99%. One challenge to formulation technologists is to minimise theselosses.

In work leading to the present invention, we have shown that the choiceof fermentation parameters can affect the eventual success of the wholeformulation process.

According to the present invention, there is provided a method ofimproving the viability of a dried bacterial cell mass comprisingmaintaining a culture of bacteria in stationary phase for a period oftime and thereafter drying the bacteria.

The period of time for which the culture should be maintained instationary phase to achieve maximum viability after drying may bedetermined by simple testing. A period the region of six hours isgenerally suitable.

Preferably also the culture is conducted in a medium deficient innitrogen.

It is also advantageous that the culture medium has a higher than normalosmotic potential.

Viability after drying may be further improved by subjecting the cultureto heat shock prior to drying. Exposure to a temperature of around 37°C. for a period of from five to 15 minutes is generally suitable.

Preferably the bacterium is a Pseudomonas, more preferably Pseudomonasfluorescens. One particular strain of interest in this invention isPseudomonas fluorescens, strain 54/96 (NCIB 40186). A culture ofPseudomonas fluorescens, strain 54/96 was deposited on Sep. 1, 1989under the terms of the Budapest Treaty with the National Collection ofIndustrial and Marine Bacteria Limited, 23 St. Marcer Road, Aberdeen AB2lRY United Kingdom, under the Accession Number 40186.

The production process of the invention pre-conditions the BCA towithstand drying processes that are an integral part of formulation ofthe product. The enhanced viability of the BCA improves the efficiencyand effectiveness of the formulation process.

One or more of the following fermentation parameters are required forpreconditioning the cells of the biological control agent:

(1) GROWTH PHASE

Drying survival depends on culture age. Maximum survival is achievedwith stationary phase cultures (i.e. when the cells are starved of somenutrient). There is a dramatic improvement in survival at the criticalpoint when cells enter stationary phase and an optimum survival aftersome period within the stationary phase (for example, at around 6 hoursinto stationary phase).

(2) NUTRIENT STARVATION

Nitrogen starved cells survive drying better than carbon starved cells.

(3) HEAT SHOCK

Cultures subjected to a short period of heat shock immediately prior todrying survive better. This increase in survival is particularly markedin log phase cultures but also occurs in stationary phase cultures.

(4) OSMOADAPTATION

Cells grown in media of high osmotic potential (for example, TSB withadded NaCl, 0.5M) are shown to accumulate the sugar trehalose. Trehaloseis known to have a protectant effect against desiccation and heat damagein many biological systems. Cells grown in this way have improvedthermotolerance which may give an advantage in a drying process.

It is possible to apply one of more of the treatments listed in (1) to(4) above synergistically in a fermentation process to increase dryingsurvival. For example, the BCA may be grown in a nitrogen-limited mediumuntil in stationary phase (preferably well into stationary phase, forexample six hours) and then heat shocked for 5-15 minutes prior todrying: this should give maximum drying survival.

By way of example only, we describe a process for production byfermentation of a Pseudomonas fluorescens strain for use as a biologicalcontrol agent (BCA). The following Examples use the biological controlagent Pseudomonas fluorescens strain 54/96 (NCIMB 40186) as described inInternational Application Publication Number WO91/05475.

Experiments investigating the effect of fermentation parameters havebeen carried out. Cells were grown in fermenters on tryptone soya broth(TSB) with and without various defined supplements and subjected tosublethal stresses before air drying on glass beads.

All experiments were done using laboratory scale (131 volume)fermentation. The base medium was Tryptone Soya Broth (TSB). TSBcontains in 1 liter: 17 g tryptone (pancreatic digest of casein); 3 gsoytone (papaic digest of soybean meal); 2.5 g dextrose; 5 g sodiumchloride; 2.5 g dipotassium phosphate. TSB (C-limited) was supplementedwith various media components to achieve increased drying survival.

Drying survival was measured primarily using a glass bead test, althoughthis is not a commercial process. Some data is presented on the effectof preconditioning using nitrogen starvation on the survival of Pfluorescens in a laboratory scale spray dryer.

Stationary phase cells survived better than growth phase cells; a sharpincrease was observed at the transition point with optimum survival sixhours into stationary phase.

Stationary phase cells grown in N-limiting media showed 5-20 fold bettersurvival than stationary phase cells grown in TSB which is C-limiting(both on the bead test and in a laboratory spray dryer).

Short periods of heat shock (37° C. for 5-10 minutes) resulted inenhanced survival although this was more pronounced in the otherwisesusceptible log phase cultures (95 fold increase) than in stationaryphase cultures (3 fold increase).

These data suggest that a fermentation process giving maximum dryingsurvival would involve growth of P fluorescens in a medium of TSBsupplemented for nitrogen limitation (recipe below) until 6 hours intostationary phase followed by heat shock for 5-10 minutes prior todrying.

It will be apparent to one skilled in the art that the processesdescribed herein by way of example must be scaled-up for use in thecommercial production of BCAs. This will involve a certain degree ofoptimisation to determine the exact parameters suitable for large-scalefermentation. However, the general principles of the invention(conditions or treatments which cause physiological adaptation such thatcells are more able to withstand drying) are clearly applicable tosmall- or large-scale fermentation.

In a preferred embodiment, therefore, the present invention provides amethod of producing a dried viable cell mass of Pseudomonas fluorescenscomprising culturing the said P. fluorescens in stationary phase in anitrogen-deficient medium, isolating the cultured cells and drying same.Preferably the culture is subjected to heat shock prior to drying.

The invention will now be described by way of example only withreference to the following drawings, wherein:

FIG. 1 is the growth curve of Pseudomonas fluorescens strain 54/96;

FIGS. 2A and 2B are graphs showing the intracellular accumulation oftrehalose in TSB culture and TSB culture amended with 0.5M NaClrespectively;

FIG. 3 shows the drying survival in N-limited media.

FIG. 4 is a graph illustrating the effect of trehalose on the stabilityof formulations.

EXAMPLE 1 DRYING AND CELL VIABILITY

Cell samples were dried by coating onto glass beads in a dish which wasleft static for up to 5 days at 20° C. Beads were sampled over a 5 dayperiod, washed in sterile distilled water (SDW), diluted serially, andplated on Tryptone soya agar (TSA) using spread plate or Spiral Plater.Plates were incubated in the dark at 28° C. for 24 h.

EXAMPLE 2 FERMENTATION

Fermenters used were Braun Biostat E, with a total volume of 18.3 l.Standard fermenter parameters were: Temperature controlled at 25° C.;Dissolved Oxygen Tension (DOT) controlled at 50% by stirrer speed andvalve setting; pH controlled at 7.0 using 2M H₂ SO₄ and 4M NaOH;antifoam controlled by additions of polypropylene glycol MW2025 (PPG2025). Medium was Tryptone Soya Broth (TSB) except where stated. Workingvolume was 13 l and a 24 h shake flask culture was used to inoculate at0.1%.

2.1 GROWTH

Biomass was routinely measured as optical density at 550 nm wavelength(OD₅₅₀)in a Corning 258 spectrophotometer. The growth curve in a BraunBiostat was defined (FIG. 1): maximum specific growth rate (μ_(max))estimated as 0.87; Doubling time(t_(d))=47 m; Growth yield=12 ODs.

2.2 BCA ACTIVITY

BCA activity of fermenter grown culture applied as a drench was comparedto shake flask culture in a standard pot test for BCA activity. 11 h(log phase) and 35 h (stationary phase) fermenter cultures plus 24 hshake flask cultures were compared. No significant difference in controlof Pythium ultimum was observed.

EXAMPLE 3 TREHALOSE ACCUMULATION AND LOSS

Cell samples were extracted for sugar analysis. Shake flask samples werespun down (15 mins, 3,000 rpm, 4° C. in Sorval RT6000B) washed in anisotonic NaCl solution spun again and re-suspended in 70% ethanol. Thissuspension was sonicated (24 microns in a Soniprep 150) until total celldisruption and the cellular matter removed by centrifugation.

Analysis of intracellular extracts was by HPLC. Samples were deionisedon Amberlite MB-3, dried by vacuum centrifugation (3,000 rpm, overnightin a Uniscience Univap). Dried samples were resuspended in deionisedwater filtered through 0.45 μm PVDF filters (Gelman Acrodisc).

Samples were analysed by HPLC (Waters Differential Refractometer SugarAnalyzer). Samples were run through filtered deionised water, with orwithout 5 mg/l Ca²⁺ EDTA, at 80° C. with a flow rate of 0.5 ml/min. Thecolumn was an Aminex HPX-42C (Biorad) and peak integration was on an IBMVG Data Systems-Minichrom v1.62)

The predominant intracellular sugar was trehalose. In TSB culturestrehalose accumulated to a maximum of 2% of cell dry weight. In TSBamended with 0.5M NaCl trehalose accumulated to 11% of cell dry weight.Maximum trehalose content was found towards the end of the growth phasewith a decline during stationary and decline phase (FIGS. 2A and 2B).

Cells harvested from TSB+NaCl were resuspended in either water orisotonic NaCl solution. At intervals after resuspension cells wererapidly separated by centrifugation and both cells and supernatantanalysed for trehalose. Cells washed in deionised water rapidly losttrehalose to the supernatant.

3.4. THERMOTOLERANCE OF TREHALOSE ACCUMULATING CELLS

Cell samples grown under osmotic stress were incubated at 50° C. beforeand after washing in sterile distilled water. Unwashed `stressed` cellsdisplayed greater thermotolerance than washed `stressed` cells. It isnot clear from the current data whether stressed cells are significantlymore thermotolerant than unstressed cells.

Cells cultured to accumulate trehalose retained their via ability betterin an accelerated shelf-life study.

Cells were grown in TSB with and without NaCl. These cells were vacuumdried in the presence of formulation additives and varying concentrationof NaCl. Samples of formulated material were stored at 37° C. and testedfor viability over a period of 76 days.

Referring to FIG. 4, samples formulated using cells grown in TSB (nos.1, 2 and 3) showed a more rapid decrease in viability over 76 days.Samples formulated using cells grown in TSB+0.25M NaCl (7, 8 and 9) toaccumulate trehalose showed a slower decrease in viability. Samples 1,4and 7 were formulated with additives in water: samples 2, 5 and 8 wereformulated with additives in 0.25M NaCl: samples 3, 6 and 9 wereformulated in 0.5M NaCl.

EXAMPLE 4 HEAT SHOCK 4.1 TEMPERATURE PROFILE

Growth rates were determined over a temperature range of 20° C. to 37°C. in TSB shake flasks. Optimum growth temperature was estimated at28°-29° C. (μ=0.72). Growth rate at 37° C. was slow (μ=0.13); Pfluorescens is known not to grow at 41° C., a diagnostic characteristic.Rapid cell death occurs at 50° C.

4.2 HEAT TREATMENT OF GROWING CELLS

Fermenter cultures were allowed to grow to late log phase at 21° C. Thetemperature was raised rapidly (-3° C. min⁻¹) to 37° C. and held there.Samples were taken immediately prior to the temperature shift and atintervals throughout. Samples were tested for drying survival.

Heat-shocked samples showed increases in survival up to 95× thepre-shock sample. The effect of heat-shock duration under theseconditions has been examined in a number of experiments. The data showsan optimum exposure of 10-15 mins with some variation betweenexperiments. There is evidence that the more severe the drying (i.e.longer incubation on the beads and hence lower moisture content) thesmaller the effect. There is also some evidence to suggest that thisdecline in effect can be off-set to some extent by increasing the heatexposure.

4.3 HEAT TREATMENT OF STARVED (STATIONARY PHASE) CELLS

Cells in stationary phase survive drying much better than those in thegrowth phase; this will be dealt with below. Thus stationary phasecultures were heat shocked and tested for drying survival. Theconditions were the same as in 4.2 except the cultures were 20-24 h old(a few hours into stationary phase).

Some reproducible but limited increase in survival could be achievedusing short periods of heat exposure (5-10 mins) with mild drying (24h). The maximum effect was a 3-fold increase. This effect is reducedwith longer drying times. Also, longer exposure to the heat-shock has adetrimental effect on survival.

4.4 GEL ANALYSIS OF HEAT SHOCK PROTEINS(HSPs)

Cell samples from the heat-shock experiments were analysed forintracellular protein content by polyacrylamide gel electrophoresis(PAGE).

At least 6 different proteins were shown to accumulate during heat-shockof growing cells. There was no detectable protein accumulation inheat-shocked stationary phase cells.

EXAMPLE 5 STARVATION 5.1 COMPLEX MEDIA

Drying survival was related to growth phase of cultures growing on TSBin fermenters. Samples taken at time intervals during the fermentationwere subjected to the bead test. There was a dramatic increase insurvival rate at the transition between growth phase and stationaryphase. Optimum survival occurred 6 hours into stationary phase (FIG. 3).

5.2 NITROGEN LIMITATION

Growth of P fluorescens in TSB is carbon limiting (C-limiting) i.e.stationary phase cells are starved of carbon. TSB was supplemented withvarying concentrations of glucose, KH₂ PO₄ and MgSO₄ in a constant ratioof 25:5:1 in an attempt to achieve N-starvation. Supplementation with 20g/l glucose showed clear increases (2/3×) in bead test survival at allincubation times over a TSB control. Cultures were not apparentlyN-limited (no increase in OD after addition of NH₄ Cl).

With the higher levels of supplementation (40 g/l glucose), in theBiolab fermenters, N-limitation was achieved with a concomitant 5-8 foldincrease in survival over the control (PS/073,074). An attempt to repeatthis in Biostat fermenters failed to show an increase in survival on thebead test. This material was also spray dried with, again, no differencefrom the control (PS/091,092). Problems with the N-limited fermentationwere identified as a possible cause of failure including temporary fallin pH to pH6, temporary fall in DOT and addition of excessive antifoam.

Confirmation of this result was achieved in the 20 liter fermentersDrying survival on beads was clearly better with the N-limited culture.The data suggests improvements in % survival of 5-20 (FIG. 3) foldalthough the variability was high. Harvest time was more clearly definedthan in previous experiments and was estimated at 4-5 hours intostationary phase for both fermentations. NB: it was more difficult toestimate this in the supplemented culture because the transition fromgrowth to stationary was more gradual--this is typical.

Washed and unwashed samples were spray dried. There was no significantdifference in total CFUs recovered. However approximately 65% ofunwashed N-limited cells and 35% of the washed was lost in the spraydryer due to sticking. Cell viability in terms of CFUs/g powder from 20g wet weight starting material was of the order of 3-fold more for theN-limited samples. However recovery was low, although there was someevidence that this was improved by washing in SDW.

                  TABLE 1                                                         ______________________________________                                                         Product                                                                             Viability                                                                              Recovery                                      Fermenter Treatment    (CFU/g)  (grams)                                       ______________________________________                                        C-limited Unwashed     1.1 × 10.sup.10                                                                  20.5                                                    Washed       2.7 c 10.sup.10                                                                        17.9                                          N-limited Unwashed     7.3 × 10.sup.10                                                                   8.5                                                    Washed       5.6 × 10.sup.10                                                                  13.2                                          ______________________________________                                    

I claim:
 1. A method for improving the viability of a dried bacterialcell mass consisting of Pseudomonas fluorescens bacteria, the methodcomprising the steps of:a) culturing the Pseudomonas fluorescensbacteria in a nitrogen deficient medium having an osmotic potentialwhereby trehalose accumulates in the cells to protect the cells againstdesiccation and heat damage; b) maintaining the bacteria in a stationaryphase for at least six hours; and c) drying the bacteria.
 2. The methodof claim 1, in which the bacterium is Pseudomonas fluorescens, strain54/96 (NCIB 40186).
 3. The method of claim 1 in which the culturing ofstep (a) is conducted in a medium consisting essentially of tryptonesoya broth containing 0.5M sodium chloride.
 4. The method of claim 1which comprises the additional step of subjecting the bacteria to heatshock prior to drying.
 5. The method of claim 4, in which the said heatshock consists of holding the culture at a temperature of around 37° C.for a period of from five to 15 minutes.
 6. The method of claim 1 inwhich the medium comprises tryptone soya broth.
 7. The method of claim 6in which the medium further comprises sodium chloride.
 8. A method forimproving the viability of a dried bacterial cell mass consisting ofPseudomonas fluorescens bacteria, the method comprising the steps of:a)culturing the Pseudomonas fluorescens bacteria in a medium having anosmotic potential whereby trehalose accumulates in the cells to protectthe cells against desiccation and heat damage; b) maintaining thebacteria in a stationary phase for at least six hours; c) subjecting thebacteria to heat shock; and d) drying the bacteria.
 9. The method ofclaim 8 in which the culturing is conducted in a medium consistingessentially of tryptone soya broth containing 0.5M sodium chloride. 10.The method of claim 8 in which said heat shock consists of holding theculture at a temperature of around 37° C. for a period of from 5 to 15minutes.
 11. The method of claim 8 in which the bacterium is Pseudomonasfluorescens, strain 54/96 (NCIB 40186).
 12. The method of claim 8 inwhich the medium comprises tryptone soya broth.
 13. The method of claim12 in which the medium further comprises sodium chloride.